Process for biological preparation of amides

ABSTRACT

In a process for hydrating a nitrile having 2 to 4 carbon atoms by a microbiological treatment to convert the nitrile into the corresponding amide, it is possible to biologically prepare the corresponding amide effectively by utilizing a microorganism of the genus Pseudomonas which is capable of hydrating the nitrile to convert the same into the corresponding amide. In particular, it is possible to obtain acrylamides which are highly useful polymers by the very simple and energy-saving process in accordance with the present invention.

TECHNICAL FIELD

The present invention relates generally to a process for hydrating loweraliphatic nitriles by a microbiological treatment to prepare thecorresponding amides. More particularly, it relates to a process forbiological preparation of amides characterized by the microorganismsused.

BACKGROUND ART

Lower aliphatic amides, for example, acrylamide, can be prepared byhydrating the corresponding nitriles, for example, acrylonitrile, and amethod in which microorganisms are utilized for the hydration has beenproposed, for instance, in Japanese Patent Laid-Open Publication No.86186/1976 and Japanese Patent Publication No. 17918/1981.

DISCLOSURE OF THE INVENTION Summary

The present invention relates to a process for biologically hydratinglower aliphatic nitriles by means of microorganisms of the genusPseudomonas to prepare the corresponding amides.

Therefore, the process for biological preparation of amides according tothe present invention which comprises hydrating nitriles having 2 to 4carbon atoms by a microbiological treatment to convert the nitriles intothe corresponding amides is characterized in that the microorganismsused are of the genus Pseudomonas and are capable of hydrating thenitriles to convert the same into the corresponding amides.

Meritorious effect

As will be noted from the examples which will be set forth hereinafter,the microorganisms used in the present invention have particularlyremarkable acrylamide-forming ability and yet show substantially nocapability of hydrolyzing acrylamide, which has once been formed, intoacrylic acid. Thus, the solution obtained by the hydration reactioncontains acrylamide in high concentration, whereby the recovery ofacrylamide can be facilitated.

Further, the optimum temperature for the hydration reaction isordinarily as low as 0° to 20° C., so that the process of this inventionis advantageous from the viewpoint of economy of thermal energy.

DETAILED DESCRIPTION OF THE INVENTION

1. Nitriles To Be Treated

Nitriles to be hydrated in accordance with the present invention arethose having 2 to 4 carbon atoms. Examples of such nitriles areacetonitrile, propionitrile, acrylonitrile, methacrylonitrile,n-butyronitrile, and isobutyronitrile. Among these nitriles,acrylonitrile is typical and can be hydrated with good results.

In the presence of the microorganisms to be used in this invention,nitriles having 5 or more carbon atoms or aromatic nitriles tend to beless easily hydrated than nitriles having 2 to 4 carbon atoms.

2. Biological Hydration Reaction

The microbiological hydration reaction of nitriles according to thepresent invention is not substantially different from the prior artmethods mentioned hereinbefore except that specific species ofmicroorganisms are used.

The term "hydration of nitriles by a microbiological treatment toconvert the nitriles into the corresponding amides" as used herein isintended to cover both the case wherein microorganisms are cultured inthe presence of nitriles and the case wherein nitriles are contactedwith cultures obtained by culturing microorganisms, cells collected fromthe cultures, or cells which have been subjected to some treatment (suchas ground cells or enzymes separated and extracted from cells). Thisterm also refers to the case wherein cells or enzymes produced therebyare immobilized and utilized in the hydration reaction. In thisconnection, the biological hydration reaction of the present inventionis considered to proceed by the aid of an enzyme (nitrile hydratase)produced by the microorganisms.

The culture of the microorganisms used in the present invention can becarried out by any method suitable for the desired purpose. For theculture media to be utilized, in general, those containing carbonsources such as glucose, maltose, and dextrins, nitrogen sources such asammonium sulfate, ammonium chloride, and nitriles other than those to behydrated, for example, isobutyronitrile in the case where acrylonitrileis hydrated, organic nutriment sources such as yeast extract, maltextract, peptone, and a meat extract, inorganic nutriment sources suchas phosphates, magnesium, potassium, zinc, iron, and manganese, and thelike are preferred.

The pH of the culture medium is of the order of 6 to 9, preferably ofthe order of 7 to 8, and the culturing temperature is of the order of20° to 37° C., preferably about 25° to 30° C. By carrying out theculture aerobically under these conditions for about 2 to 3 days,sufficiently active cells can be produced in sufficient quantity.

The culture may be carried out in the presence of a nitrile to behydrated (the nitrile concentration being about 0.5 to 2% by weight ofthe culture) as has been set forth earlier, but the hydration reactionis preferably accomplished by isolating cells from the culture (forexample, by centrifugation), dispersing the cells in water, isotonicsodium chloride solution or like aqueous media, and adding thereto anitrile to be hydrated.

In this preferred mode of the invention, the hydration of a nitrile isaccomplished by reacting an aqueous dispersion containing 0.1 to 5% byweight of cells of a microorganism and 0.5 to 2% by weight of a nitrileat a temperature of the order of 0° to 20° C., preferably 0° to 5° C.,and a pH of about 6 to 9, preferably 7 to 8, for a period of the orderof 1 to 30 hours. It is desirable that the consumed nitrile besupplemented continuously or intermittently so that the nitrileconcentration in the reaction solution will be maintained within therange specified above. In order to maintain the pH value within thespecified range, it is preferable to employ an appropriate buffersystem. The microorganisms used in the present invention have a poorcapability to further hydrate the amides produced, but, when the pH ofthe reaction system becomes excessively acidic or alkaline, hydrolysisproceeds so that carboxylic acids, for instance, acrylic acid, tend tobe formed. The reaction temperature in the above specified range isgenerally suitable, but temperatures not exceeding 5° C. are preferredin the case where the hydration reaction is carried out over a longperiod of time, e.g., for 5 hours or longer with the addition ofnitrile.

After hydration is conducted for some period of time, the nitrile isconverted into the corresponding amide with a conversion of nearly 100%which is accumulated in the reaction solution in a high concentration.The amide can be collected from the reaction solution in accordance withany suitable method. For example, by separating cells from the reactionsolution by centrifugation or like procedure, removing stainedsubstances and impurities by treatment with activated carbon, ionexchange resins and the like, and then concentrating the reactionsolution under reduced pressure, the desired amide, for example,acrylamide, can be obtained in crystalline form.

3. Microorganisms Used

(1) Species and Mycological Properties

The microorganisms used in the present invention are bacteria of thegenus Pseudomonas capable of producing amides through hydration of thenitriles mentioned previously.

Specific examples of the strains of such microorganisms are Pseudomonaschlororaphis B 23 (hereinafter abbreviated to B 23) and Pseudomonas sp.PS 1 (hereinafter abbreviated to PS 1).

The principal mycological properties of these microorganisms are asfollows.

    ______________________________________                     B 23          PS 1    ______________________________________    (a)  Morphology    1    Shape and size                     bacillus      bacillus         of cell     0.8 - 1.1 × 1.6 -                                   0.8 - 1.1 × 1.3 -                     2.7 μm     1.9 μm    2    Polymorphism                     none          none    3    Motility    motile        motile                     one to three  with polar fla-                     polar flagella                                   gella    4    Formation of                     none          none         spores    5    Gram staining                     -             -    6    Acid-fast   -             -         property    (b)  Growth on         various culture         media    1    Bouillon-agar                     spherical,    smooth, homoge-         plate culture                     convex, glossy,                                   neous, glossy,                     translucent,  and mucoidal                     and yellow    2    Bouillon-agar                     small colony  smooth, glossy,         slant culture                     formed        translucent,                                   and yellow    3    Bouillon liquid                     precipitated         culture    4    Bouillon-gela-                     liquified (+) -         tin stab         culture    5    Litmus-milk acidic: pepto-                                   alkaline: pepto-                     nized, not    nized, not                     coagulated    coagulated    (c)  Physiological         properties    1    Reduction of                     +             -         nitrate    2    Denitrifica-                     +             -         tion    3    MR test     -             -    4    VP test     -             -    5    Formation of                     -             -         indole    6    Formation of                     -             -         hydrogen         sulfide    7    Hydrolysis of                     -             -         starch    8    Utilization of                     Simon's cul-  Simon's cul-         citric acid ture: +       ture: +    9    Utilization of                     ammonium salt:                                   ammonium salt:         inorganic   +             +         nitrogen         source    10   Formation of                     King-A culture:                                   King-A culture:         pigments    -             -                     King-B culture:                                   King-B culture:                     +             +                     green (water- green (water-                     soluble)      soluble)    11   Urease      -             -    12   Oxydase     +             +    13   Catalase    +             +    14   Growth range                     pH: 6.0-9.9                     temperature:                     5-36.5° C.    15   Behavior    aerobic       aerobic         toward oxygen    16   O-F Test    oxidized      oxidized    17   Formation of                     Formation                              Formation                                     Formation                                            Formation         acid & gas  of acid  of gas of acid                                            of gas         from         saccharide         D-glucose   +        -      +      -         D-mannose   +        -      +      -         D-fructose  -        -      -      -         D-galactose +        -      +      -         maltose     -        -      -      -         sucrose     -        -      -      -         lactose     -        -      -      -         trehalose                   -      -         D-mannitol  -        -      -      -         glycerol    -        -      -      -         starch      -        -      -      -    18   Nutritive re-                     none          none         quirements    19   Other proper-                     See remarks         ties    ______________________________________    Remarks: Aminopeptidase        +             Formation of levan from                                   +             saccharose             Formation of poly-β-hydroxy-                                   -             butyrate             GC content            64.6%

(2) Deposit

The microorganism listed above have been deposited with the FermentationResearch Institute, Agency of Industrial Science & Technology under thefollowing numbers.

    ______________________________________    Strain    Deposit No.   Deposit Date    ______________________________________    B 23      FERM BP-187   November 16, 1981    PS 1      FERM BP-188   November 16, 1981    ______________________________________

The deposit of these microorganisms was made in conformity with the"BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OFMICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE". The aforesaidFermentation Research Institute, Agency of Industrial Science andTechnology, which is entitled to an international depository authority,is situated at 1-3, Higashi 1 chome, Yatabe-machi, Tsukuba-gun,Ibaraki-ken, Japan (postal no.: 305).

BEST MODE OF PRACTICE OF THE INVENTION

Hereinafter, the process for biological preparation of amides accordingto the present invention will be specifically described in concreteterms with respect to examples of practice thereof.

EXAMPLE 1

(1) Culture

Strain B 23 was cultured under the following conditions.

    ______________________________________    (1)     Culture Medium            Dextrin              0.5%            K.sub.2 HPO.sub.4    0.2%            MgSO.sub.4.7H.sub.2 O                                 0.02%            Isobutyronitrile     0.2%            NaCl                 0.1%            pH                   7.0    (2)     Cultural Conditions: 28° C./3 days    ______________________________________

(2) Hydration of Acrylonitrile

Acrylonitrile was added intermittently to the thus obtained culture,which was utilized as an enzyme source without treatment, for hydrationthereof.

The reaction was carried out at 0° to 4° C. from the time whenacrylonitrile was added, and the addition of acrylonitrile was continuedto cause the concentration of acrylonitrile to be 0.4 M.

About 100 g/l of acrylonitrile was produced 84 hours after theinitiation of the reaction. The acrylamide yield was nearly 99%, and thequantity of acrylic acid formed as a by-product was only 0.4% of theacrylonitrile added.

The isolation of acrylamide from the reaction solution was accomplishedby freeze-drying the solution, extracting the freeze-dried substancewith methanol, and concentrating the methanol extract to form aprecipitate of crystals. The crystals obtained were recrystallized frommethanol, and colorless, sheet-like crystals were obtained andidentified as acrylamide from the melting point and by elementaryanalysis, NMR an IR.

EXAMPLE 2

(1) Culture

The procedure of Example 1 was followed.

(2) Hydration of Acrylonitrile

Cells were separated from the culture obtained, washed with water, anddispersed in water (potassium phosphate buffer pH 7.0) in a quantitysufficient to reach a concentration of 20 mg/ml, based on the dry weightof the cells, to form a dormant cell dispersion to which acrylonitrilewas added continuously for hydration thereof.

The reaction was carried out at 0° to 4° C., and acrylonitrile was addedintermittently to cause the concentration of acrylonitrile to be 0.4 M.

200 g/l of acrylamide was produced substantially linearly 7.5 hoursafter the initiation of the reaction. The reaction seemed to proceedfurther but was terminated at this stage since the reaction solutionbecame viscous.

The acrylamide yield was nearly 99%, and the quantity of acrylic acidformed as a by-product was only about 0.7% of the acrylonitrile added.

The acrylamide thus produced was collected and identified as such in thesame manner as in Example 1.

EXAMPLE 3

The substrate specificity of a nitrile hydratase in strain B 23 wasexamined by the cell method.

(1) Culture of Cells

The procedure of Example 1 was followed.

(2) Hydration of Nitrile

Cells were separated from the culture obtained, washed with water, andsubjected to reaction under the following conditions. The volume of thereaction solution was 1 ml.

    ______________________________________    Substrate nitrile   300 μmole    Potassium phosphate buffer                        pH 7.0/100 μmole    Cell                10 mg (as dry                        cell)    Temperature         30° C.    Reaction time       1 hour    ______________________________________

The results obtained were as tabulated below.

    ______________________________________                  Amide-Forming Activity    Test Nitrile    (unit/mg)* (%)    ______________________________________    Acetonitrile    0.11       31    Propionitrile   0.41       117    Acrylonitrile   0.35       100    Methacrylonitrile                    0.08       23    n-Butyronitrile 0.20       57    Isobutyronitrile                    0.18       51    ______________________________________     *unit/mg: The activity which enables 1 mg of a cell to form 1     μmole/min. of acrylamide was defined as "1 unit".

EXAMPLE 4

This example shows a comparison of the hydration reactions of strains B23 and PS 1.

(1) Culture of Cells

Cells were cultured similarly as in Example 1 except that 0.01% of yeastextract was further added to the culture medium of Example 1.

(2) Hydration of Acrylonitrile

Cells were separated from the culture obtained, washed with water, andsubjected to reaction under the following conditions. The volume of thereaction solution was 1 ml.

    ______________________________________    Acrylonitrile       300 μmole    Potassium phosphate buffer                        pH 7.0/100 μmole    Cell                10 mg (as dry cell)    Temperature         15° C.    Reaction time       2 and 4 hours    ______________________________________

The results obtained were as tabulated below.

    ______________________________________                Formation of Acrylamide (%)    Strain        2 hours   4 hours    ______________________________________    B 23          90        100    PS 1          82        97    ______________________________________

INDUSTRIAL APPLICABILITY

As has been stated above, the present invention, which is directed to aprocess for biological preparation of amides, provides a process forhydrating lower aliphatic nitriles by using specific microorganisms ofthe genus Pseudomonas to effectively convert the nitriles into thecorresponding amides. Especially, the present invention ensures a verysimple and energy-saving process for preparing acrylamides which arehighly useful polymers.

We claim:
 1. A process for hydrating a nitrile having 2-4 carbon atomsto the corresponding amide which comprises contacting a nitrile with amicroorganism of the genus Pseudomonas which produces nitrile hydratase.2. The process as claimed in claim 1, wherein the nitrile is added to aculture of the microorganism in order to convert the nitrile into thecorresponding amide.
 3. The process as claimed in claim 1, wherein thenitrile is added to an aqueous dispersion of cells separated from aculture of the microorganism in order to convert the nitrile into thecorresponding amide.
 4. The process as claimed in either claim 2 orclaim 3, wherein the nitrile is added continuously or intermittently. 5.The process as claimed in claim 1, wherein the nitrile is acrylonitrileand the microorganism is Pseudomonas chlororaphis B 23 (FERM BP-187) orPseudomonas sp. PS 1 (FERM BP-188).
 6. The process as claimed in claim5, wherein the microorganism is Pseudomonas chlororaphis B
 23. 7. Theprocess as claimed in claim 1, wherein nitrile consumed during thehydration is supplemented during the reaction so that the nitrileconcentration is maintained within the range of about 0.5 to 2% byweight.
 8. The process as claimed in claim 7, wherein the nitrile isacrylonitrile.
 9. The process as claimed in claim 1 wherein thehydration is carried out at a pH of about 6 to 9 and substantially nohydrolysis of the amide to an acid takes place during the hydration. 10.The process as claimed in claim 1 wherein the process is carried out ata temperature of 0° to 20° C.
 11. A process for the biologicalpreparation of an amide having 2 to 4 carbon atoms by hydrating thecorresponding nitrile which comprises treating the nitrile with amicroorganism selected from the group consisting of Pseudomonaschlororaphis B 23 (FERM BP-187) and Pseudomonas sp. PS 1 (FERM BP-188).12. The process as claimed in claim 11, wherein the nitrile isacrylonitrile.
 13. The process as claimed in claim 11 wherein themicroorganism is Pseudomonas chloraphis B
 23. 14. A process forhydrating a nitrile having 2-4 carbon atoms to the corresponding amidewhich comprises contacting a nitrile with nitrile hydratase produced bya microorganism of the genus Pseudomonas.
 15. The process as claimed inclaim 14, wherein the nitrile is added continuously or intermittently.16. The process as claimed in claim 14, wherein nitrile consumed duringthe hydration is supplemented during the reaction so that the nitrileconcentration is maintained within the range of about 0.5 to 2% byweight.
 17. The process as claimed in claim 14, wherein the hydration iscarried out at a pH of about 6 to 9 and substantially no hydrolysis ofthe amide to an acid takes place during the hydration.
 18. The processas claimed in claim 14 wherein acrylonitrile is converted to acrylamide.19. The process as claimed in claim 14, wherein the process is carriedout at a temperature of 0° to 20° C.
 20. The process as claimed in claim14 wherein the microorganism is Pseudomonas chlororaphis B 23 (FERMBP-187) or Pseudomonas sp. PS 1 (FERM BP-188).
 21. A method for thepreparation of acrylamide from acrylonitrile comprising contactingacrylonitrile with nitrile hydratase produced by a microorganism of thegenus Pseudomonas.